U.S. patent application number 10/482235 was filed with the patent office on 2004-12-09 for methods for purification of bacterial cells and components.
Invention is credited to Frick, Sibylle, Grassl, Renate, Meyer, Roman, Miller, Stefan, Robl, Ingrid, Schutz, Michael, Zander, Thomas.
Application Number | 20040248298 10/482235 |
Document ID | / |
Family ID | 7688882 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248298 |
Kind Code |
A1 |
Schutz, Michael ; et
al. |
December 9, 2004 |
Methods for purification of bacterial cells and components
Abstract
The present invention relates to a method for the selective
purification of bacterial cells and/or cell components, whereby the
purification is performed by means of a solid support.
Inventors: |
Schutz, Michael;
(Lappersdorf, DE) ; Grassl, Renate; (Regensburg,
DE) ; Meyer, Roman; (Schmidmuhlen, DE) ;
Frick, Sibylle; (Zeitlarn, DE) ; Robl, Ingrid;
(Regensburg, DE) ; Zander, Thomas; (Lappersdorf,
DE) ; Miller, Stefan; (Regensburg, DE) |
Correspondence
Address: |
Steven L Highlander
Fulbright & Jaworski
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Family ID: |
7688882 |
Appl. No.: |
10/482235 |
Filed: |
June 15, 2004 |
PCT Filed: |
June 24, 2002 |
PCT NO: |
PCT/DE02/02302 |
Current U.S.
Class: |
435/383 |
Current CPC
Class: |
C12N 2795/10222
20130101; C12N 15/1013 20130101; Y10T 436/10 20150115; Y10S 436/802
20130101; C07K 14/005 20130101 |
Class at
Publication: |
435/383 |
International
Class: |
C12N 007/00; C12N
007/01; C12N 005/00; C12N 005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2001 |
DE |
101 29 815.3 |
Claims
1. A method for the selective purification of bacterial cells or
cell components, comprising the following steps: a) contacting a
sample containing bacterial cells or cell components with
bacteriophages and/or bacteriophage proteins; b) subsequent
incubation of the sample, containing the bacterial cells or cell
components and the bacteriophages and/or bacteriophage proteins,
with a solid support, wherein the solid support exhibits one or
more different coupling group(s) on its surface binding the
bacteria and/or bacteriophage proteins; and c) separating the solid
support with the bacterial cells or cell components bound thereto
via the bacteriophages and/or bacteriophage proteins from the
sample.
2. A method according to claim 1, wherein the coupling group is a
lectin, receptor or anticalin.
3. A method according to claim 1, wherein the coupling group is a
streptavidin or Avidin and the bacteriophage proteins are coupled
with biotin or a strep-tag.
4. A method according to claim 1, wherein the solid support is a
magnetic particle, agarose particle, glass particle, luminex
particle, reaction tube, or a microtiter plate.
5. A method according to claim 1, wherein two or more different
bacteriophages and/or bacteriophage proteins are added.
6. A method for the selective purification of bacterial cells or
cell components, comprising the following steps: a) contacting a
sample containing bacterial cells and/or cell components with a
magnetic particle, on the surface of which bacteriophages and/or
bacteriophage proteins are applied; and b) separation of the
magnetic particle with the bacterial cells and/or cell components
bound thereto from the sample.
7. A magnetic particle coated with bacteriophages and/or
bacteriophage proteins.
8. A magnetic particle according to claim 7, wherein the
bacteriophages and/or the bacteriophage proteins are selected from
the group consisting of 0c1r, 10tur, L2, L51, M1, MVG51, MV-L1, O1,
SpV1, V1, V1, V2, V4, V5, 108/016, 119, 29, 37, 43, 51, 59.1,
A1-Dat, Aeh2, Bir, M1, MSP8, .o slashed.115-A, .o slashed.150A, .o
slashed.31C, P-a-1, PhiC, R1, R2, SK1, SV2, VP5, Ap3, Ap4, Mm1,
Mm3, Mm4, Mm5, phiUW 51, 43, 44RR2.8t, 65, Aeh1, PM1, PIIBNV6, PS8,
psi, PT11, 8764, A5/A6, A6, PM2, W11, W2, W4, W7, 1A, alpha, AP50,
BLE, F, G, GA-1, II, IPy-1, mor1, MP13, MP15, .o slashed.105, .o
slashed.29 (phi 29), .o slashed.NS11, PBP1, PBS1, SP10, SP15, SP3,
SP8, SPP1, SP.beta., SPy-2, SST, type,168, W23, SP50, W23, SP01,
MAC-1, MAC-2, MAC-4, MAC-5, MAC-7, Tb, .o slashed.Cb12r, .o
slashed.Cb2, .o slashed.Cb23r, .o slashed.Cb4, .o slashed.Cb5, .o
slashed.Cb8r, .o slashed.Cb9, .o slashed.CP18, .o slashed.CP2, .o
slashed.Cr14, .o slashed.Cr28, O11, P13, O2, O3, O5, O6, O8, 1,
phiCPG1, Ce.beta., F1, HM2, HM3, HM7, 7/26, A, A19, AN25S-1, Arp,
AS-1, BL3, CONX, MT, N1, .o slashed.A8010, S-6(L), .beta.,A-4(L),
AC-1, LPP-1, S-2L, S-4L, SM-1, P1, T1, TuIa, TuIb, TuII, 1.o
slashed.3, 1.o slashed.7, 1.o slashed.9, 2D/13, Ae2, alpha10,
alpha3, BE/1, BF23, dA, delta1, delta6, d.o slashed.3, d.o
slashed.4, d.o slashed.5, Ec9, eta8, f1, fd, G13, G14, G4, G6,
HK022, HK97, HR, lambda, M13, M13 mp18, M20, MM, MS2, Mu, O1, .o
slashed.80, .o slashed.A, .o slashed.R, .o slashed.X174, PA-2, P1,
P1D, P2, P22, Q.beta., R17, S13, St-1, T1, T2, T3, T4, T5, T6, T7,
WA/1, WF/1, WW/1, zeta3, ZG/2, ZJ/2, C21, omega 8, U3, chi, FC3-9,
.mu.2, 01, 11F, 121, 1412, 3, 3T+, 50, 5845, 66F, 7480b, 8893, 9,
9266, al, alpha15, b4, B6, B7, Beccles, BZ13, C-1, C16, C2, C-2,
DdVI, Esc-7-11, f2, fcan, FI, Folac, fr, GA, H, H-19J, I2-2,
1alpha, ID2, If1, If2, Ike, JP34, JP501, K19, KU1, M, M11, M12,
MS2, NL95, .o slashed.92, .o slashed.I, .O slashed.ii, Omega8,
pilHalpha, PR64FS, PRD1, PST, PTB, R, R17, R23, R34, sd, SF, SMB,
SMP2, SP, .beta., ST, tau, tf-1, TH1, TW18, TW28, ViII, VK, W31, X,
Y, ZG/1, ZIK/1, ZJ/1, ZL/3, ZS/3, AP3, C3:, 1b6, 223, fri, hv,
hw222a, .o slashed.FSW, PL-1, y5, 1, 643, c2, kh, m13, P008, P127,
1358, 1483, 936, 949, BK5-T, c2, KSY1, P001, P008, P107, P335,
PO34, PO87, pro2, 4211, psi M2 (.PSI.M2), N1, N5, Br1, C3, L3, I3,
lacticola, Leo, .o slashed.17, R1-Myb, N13, N18, N24, N26, N36, N4,
N5, X1, X10, X24, X3, X5, X6, D3, D4, 22, 32, AU, C-2, P1, P2, P3,
P4, Phi CT, phi CTX, PB-1, 12S, 7s, D3, F116, gh-1, gh-1, Kf1, M6,
.o slashed.1, .o slashed.KZ, .o slashed.W-14, Pf1, W3, 2, 16-2-12,
2, 317, 5, 7-7-7, CM1, CT4, m, NM1, NT2, .o slashed.2037/1, .o
slashed.2042, .o slashed.gal-1-R, WT1, Mp1, MP2, W1, epsilon15,
Felix 01, 16-19, 7-11, H-19J, Jersey, N4, SasL1, ViI, ZG/3A, San21,
A3, A4, P22, 4, C1/TS2, Sp1, 107, 187, 2848A, 3A, 44AHJD, 6, 77,
B11-M15, Twort, 182, 2BV, A25, A25-24, A25-omega8, A25-PE1,
A25-VD13, CP-1, Cvir, H39, P23, P26, phi A.streptomycini III,
phi8238, phiC31, S1, S2, S3, S4, S6, S7, SH10, Tb1, Tb2, Ts1,
06N-22P, 06N-58P, 06N-58P, 4996, alpha3alpha, I, II, III, IV,
kappa, nt-1, OXN-100P, OXN-52P, v6, Vf12, Vf33, VP1, VP11, VP3,
VP5, X29, Cf, Cf1t, RR66, 8/C239, phiYeO3-12, and YerA41.
9. A magnetic particle according to claim 7, having a diameter of
about 0.5 to about 4 .mu.m or about 0.8 to about 1.8 .mu.m.
10. A bacteriophage protein comprising a strep-tag or a
his-tag.
11. A bacteriophage protein according to claim 10, whereby the tag
exhibits an amino acid sequence according to SEQ ID NO: 5, 6 or
7.
12. A bacteriophage protein according to claim 10, whereby the
bacteriophage protein is the p12 protein of the phage T4.
13. A bacteriophage protein according to claim 10, whereby the
bacteriophage protein is produced via DNA recombination
technology.
14. A nucleic acid encoding a bacteriophage protein according to
claim 10.
15. An amino acid segment having a sequence according to SEQ ID NO:
6, 7 or 8.
16. A method for the isolation of nucleic acids or cell components
comprising contacting a magnetic particle of claim 7 with a
biological sample.
17. A kit for the purification of bacterial cells and/or cell
components, comprising the magnetic particles according to claim
7.
18. A kit for the purification of bacterial cells and/or cell
components, comprising the bacteriophage proteins according to
claim 10.
19. The method of claim 16, wherein said method comprises plasmid
preparation.
20. The method of claim 16, wherien said cell components comprise
lipopolysaccharides, endotoxines or exopolysaccharides.
21. A method for the isolation of nucleic acids or cell components
comprising contacting a bacteriophage protein according to claim 10
with a biological sample.
22. The method of claim 21, wherein said method comprises plasmid
preparation.
23. The method of claim 21, wherien said cell components comprise
lipopolysaccharides, endotoxines or exopolysaccharides.
Description
[0001] The present invention relates to a method for the selective
purification of bacterial cells and/or cell components wherein the
purification is performed by means of a solid support.
[0002] The starting point of almost any further processing,
analysis, or isolation of cell components is the enrichment of the
cells, named cell harvest, usually being carried out by means of
centrifugation. This centrifugation step is the main problem of the
entire automation of methods, for example of the plasmid
purification, since in addition to the high technical complexity
for the integration of a centrifuge in a respective processing
robot an extremely high precision of the start and stop position of
the centrifugation process is required. Automatic methods of the
further processing analysis or isolation of cell components usually
starts with cells being enriched, centrifuged, or sedimented
outside of the processing robot. For example, a nearly entire
automation of the relevant methods is essential for a rapid
analysis of complete genomes, proteoms, and also for a rapid
determination of the structure and function in high throughput
methods. The automation for example in the genome analysis has
already been highly advanced: The bacterial growth as well as the
plasmid isolation may be carried out automatically. However, an
entire automation of the methods including the cell harvest is
still not feasible. Particularly a selective cell harvest, that is
the specific enrichment of particular cells out of a cell mixture,
is not possible with the presently used methods.
[0003] The cell harvest is usually carried out with the following
methods: The standard method of the cell harvest is the unspecific
centrifugation of the bacterial cultures. A microplate centrifuge
is necessary especially in those methods which are constructed for
a higher throughput. However, the centrifugation as such is not
suitable for an automation.
[0004] Though the filtration of the cultivated cells with
respective filter membranes is feasible, that filtration also
allows only an unspecific enrichment of the cells. In addition, the
method is highly accident sensitive with respect to plugging in
highly enriched cell suspensions and the high viscosity of the
solutions as a consequence thereof.
[0005] The fluorescence activated cell sorting is a method in which
a very thin liquid thread is used allowing the sorting and
enrichment of single fluorescence labelled cells by means of the
laser. By use of a respective fluorescence label, a certain
specificity of the enrichment is possible, but due to the thin
liquid thread, the method is limited to small volumes and thus to a
low throughput. Therefore, only small cell amounts may be enriched,
which is not sufficient for a further processing or an analysis of
cell components. The high costs for the apparative equipment also
prevent a strong propagation of the technique and slow down the
simultaneous work necessary for a high throughput cell harvest.
[0006] The cells are bound directly to magnetic particles via ionic
interactions and are locally concentrated by applying a magnetic
field in case of the magnetic cell separation technique. Those
methods for the unspecific concentration of bacterial cells have
been distributed recently for the entire automation of the
processing of plasmid or genomic DNA including the cell harvest by
Chemagen (EP 1 118 676), Genpoint (WO 01/53525, WO 98/51693), Merck
(WO 00/29562), as well as Promega (U.S. Pat. No. 6,284,470) or
Amersham (WO 91/12079). However, these magnetic particles exhibit
the drawback that on the one hand they bind the bacterial cells in
an unspecific manner, and on the other hand they do not bind every
bacterial species equally well. Due to the unspecificity of the
binding, even different binding efficiencies in various strains of
a species have been observed (Merck WO 00/29562).
[0007] Thus, one object of the present invention is the provision
of a method which is feasible to selectively and fully
automatically enriched bacterial cells and cell components and
which may be incorporated into an automated analysis or isolation
method. A further object of the present invention is the provision
of solid supports for the selective enrichment of bacterial cells
or cell components.
[0008] The object is achieved by the subject matter defined in the
claims.
[0009] The following figures illustrate the invention.
[0010] FIG. 1 displays in a graphic the time dependency of the
p12-dependent binding of E. coli to magnetic beads. The values
indicate the .beta.-galactosidase activity of immobilised cells in
relative units. VIK (rhombus symbols) stands for: two-step method
(preincubation of cells and p12, subsequent binding to magnetic
beads). VC (squares) stands for: one-step method (precoating of p12
to magnetic beads, subsequent immobilising of cells). -p12
(triangles) stands for: background (unspecific cell binding to
beads without p12).
[0011] FIG. 2 displays in a graphic the binding of E. coli to
magnetic beads via the bacteriophage T4. The values indicate the
.beta.-galactosidase activity of immobilised cells in relative
units. VIK stands for: two-step method (preincubation of cells and
T4, subsequent binding to magnetic beads). VC stands for: one-step
method (precoating of T4 to magnetic beads, subsequent immobilising
of cells). K stands for: background (unspecific cell binding to
beads without T4).
[0012] FIG. 3 displays in a graphic comparison the yield of E. coli
cells from different media. -p12 denotes the results without
N-strep-p12. +p12 denotes the results with N-strep-p12. Hatched
bars show the results with the strain E. coli LE392, filled in bars
denote the results with the strain E. coli JM83. LB, SOB, SOC, TB,
and YT 2.times. denote the respective media being used in the
experiment. The values are shown as yield in % of the used cells,
determined over the scattering of the supernatant at 600 nm after
pelleting of the bound cells by means of a magnet.
[0013] FIG. 4 shows graphically the result of the plasmid isolation
after harvest of E. coli with p12 according to the two-step method.
Strain DH10b with plasmid pUC19. 1: centrifuged cells, DNA
isolation via solid phase extraction, 2: cells harvested with the
two-step method according to the present invention, DNA isolation
via solid phase extraction, 3: centrifuged cells, DNA isolation via
magnetic beads, 4: cells harvested with the two-step method
according to the present invention, DNA isolation via magnetic
beads, 5: standard.
[0014] FIG. 5 shows in a graphic display the enrichment of E. coli
cells from 10 ml culture volume, starting from a cell suspension of
different cell densities (10.sup.9-10.sup.7CFU/ml). Graph A: the
filled in bars indicate the .beta.-galactosidase activity of the
cells bound via N-strep-p12, the hatched bars indicate the
background without p12. Graph B: the filled in bars indicate the
.beta.-galactosidase activity of cells bound via T4-bio, the
hatched bars indicate the background without T4.
[0015] FIG. 6 shows schematically the harvest of living E. coli via
biotinylated p12 and streptavidin beads. The filled in bars
indicate the scattering at 600 nm after two hours of growth. The
hatched bars indicate the .beta.-galactosidase activity of the
cells. The abbreviations stand for: P12: the determinations of the
cells immobilised to the beads, P12-EDTA: determinations of the
cells stripped from the beads after the binding (supernatant after
EDTA treatment), EDTA-p12: immobilising of the cells to the beads
was prevented by the presence of EDTA; determinations of the
unspecific cells to the beads, K: control experiment without p12;
determinations at the beads.
[0016] FIG. 7 shows in a table the selective binding of p12 to
bacterial cells. Table A lists the p12-dependent binding of
different E. coli strains, table B shows the specificity of the
p12-dependent binding. The abbreviation n.d. indicates not
determined, +indicates the binding of p12 to the named bacteria,
-indicates no binding of p12 to the cells.
[0017] FIG. 8 shows graphically the specificity of the
p12-dependent binding. The values indicate the yield of immobilised
cells, determined over the scattering in the supernatant at 600 nm.
The hatched bars indicate the values for the cell binding over
N-strep-p12 (=specific binding). The dark filled in bars show the
control without p12 (=unspecific adsorption). The light filled in
bars indicate the values for the cell binding with p12, in the
presence of 10 mM EDTA (=unspecific adsorption), however.
[0018] FIG. 9 shows images of light microscopy and fluorescence
microscopy of the selective binding of E. coli to magnetic beads
via T4-bio in a mixed culture of E. coli and Serratia marrescens.
The E. coli cells were fluorescence labelled with FITC-labelled
T4p12. The images on the left show exposures of light microscopy,
the images on the right show exposures of fluorescence microscopy.
The upper images show exposures of an experiment without T4-bio,
the lower images show exposures of an experiment with T4-bio.
[0019] FIG. 10 shows graphically the result of the cell harvest
after the two-step method with N-strep-p12 in solutions with
various cell numbers. S/N-ratio indicates the signal to noise-ratio
(signal with N-strep-p12 divided by the signal without N-strep-p12)
of the .beta.-galactosidase reaction of the bound E. coli cells,
CFU/ml indicates the used cells (cell forming units) per ml. The
squares indicate the results of the .beta.-galactosidase activity
of the measurement with a luminescent substrate. The rhombi
indicate the results of the .beta.-galactosidase activity of the
measurement with a fluorescent substrate.
[0020] FIG. 11 shows graphically the result of the harvest of E.
coli cells after the one-step method with T4p12 which is covalently
bound to magnetic EM2-100/49 beads (Merck Eurolab). +p12 indicates
the results with beads with T4p12, -p12 indicates the results with
beads without T4p12, DSMZ 613 indicates the results with the strain
E. coli DSMZ 613, DSMZ 13127 indicates the results with the strain
E. coli DSMZ 13127.
[0021] FIG. 12 shows graphically the results of the harvest of E.
coli cells after the one-step method with T4p12 adsorbed to
magnetic PVA-beads. The rhombi indicate the results with the beads
PVA-011 (Chemagen), the squares indicate the results with the beads
PVA-012. The continuous lines indicate the results with beads
having adsorbed T4p12, dashed lines indicate beads without T4p12.
The OD600 values indicate the %-values of harvested cells in % of
the scattering of the supernatant after pelleting of the bound
cells by means of a magnet.
[0022] The term "phage proteins" or "bacteriophage proteins", as
used herein, refers to all bacteriophage proteins participating in
the recognition and binding of the bacterial cells or the cell
components. Said proteins my be localised depending on the
morphological property of the phages, for example directly on the
phage coat or on specific recognition structures, namely the tail
fibres. Thus, the term "bacteriophage tail proteins" refers to
phage proteins displaying the bacteriophage tail or being a part of
the bacteriophage tail.
[0023] The term "specificity" as used herein means that the
bacteriophages or phage proteins recognise and bind only a single
genus or species, or a sub-species of bacterial cells or cell
components, as well as that some bacteriophages or phage proteins
recognise and bind specific bacteria groups.
[0024] The term "enrichment" or "purification" as used herein means
the specific separation of bacterial cells or cell components from
the aqueous solution, for example from the culture medium, in which
the bacterial cells or cell components are located. The
purification or enrichment is carried out by means of solid
supports, for example magnetic particles, glass particles, agarose
particles, reaction tubes, or microtiter plates.
[0025] One aspect of the present invention refers to the provision
of methods for selective purification of bacterial cells or cell
components, comprising the following steps: (two-step method)
[0026] a) contacting a sample containing bacterial cells or cell
components with bacteriophages and/or bacteriophage proteins,
preferably with an incubation time of about 3-5 minutes,
[0027] b) subsequent incubation of said sample, containing the
bacterial cells or cell components and the bacteriophages and/or
bacteriophage proteins with a solid support, preferably for about
3-30 minutes,
[0028] c) separation of the solid support with the bacterial cells
or cell components bound via the bacteriophages and/or
bacteriophage proteins to said solid support from the sample.
[0029] Bacterial cells or cell components may be enriched
selectively with methods according to the present invention, for
example from mixed cultures of different species or from a culture
of a single species. Enriched cell components may be, for example,
endotoxines, proteins, nucleic acids, or saccharides. The choice of
appropriate bacteriophages and/or bacteriophage proteins allows the
selectivity of the method. According to the method of the present
invention, bacteriophages and/or bacteriophage proteins are most
suited for a selective enrichment of bacteria or cell components,
because phage-bacteria-systems have been evolved in nature for a
long time so that the phages identify their host bacteria in a
highly specific manner and with high binding affinity. Preferably
bacteriophage proteins are used for the method of the present
invention which are specific for the bacteria desired to be
detected. Bacteriophages as well as bacteriophage proteins
developed under adverse environmental conditions so that they are
stable over influences, like temperature and pH-variations (Burda
et al., Biological Chemistry 2000, 381, 255-258) et al., and thus
may be used in the different purification buffers.
[0030] Which bacteriophages and/or bacteriophage proteins will be
used depends on the fact which bacteria species are to be purified.
For a following plasmid purification, those bacteriophages and/or
bacteriophage proteins will be preferred which may bind the E. coli
bacteria selectively, because they represent the commonly used
bacteria for a plasmid preparation at present. A large number of
known bacteriophages is available already for most of the bacteria
described so far and may be utilised for a selective bacteria
enrichment. The following table shows an overview of bacteria and
their specific bacteriophages without being exhaustive. The phages
and their respective host bacteria are commercially available from
the following strain collections: ATCC (USA), DSMZ (Germany), UKNCC
(Great Britain), NCCB (Netherlands), and MAFF (Japan). Moreover,
bacteriophages directed against respective bacteria may be isolated
for example from environmental samples according to standard
methods, if required (Seeley, N. D. & Primrose, S. B., 1982, J.
Appl. Bacteriol. 53, 1-17).
1 Bacteria: Phage Acholeplasma: 0c1r, 10tur, L2, L51, M1, MVG51,
MV-L1, O1, SpV1, V1, V1, V2, V4, V5 Actinomycetes: 108/016, 119,
29, 37, 43, 51, 59.1, A1-Dat, Aeh2, Bir, M1, MSP8, .o
slashed.115-A, .o slashed.150A, .o slashed.31C, P-a-1, PhiC, R1,
R2, SK1, SV2, VP5 Actinoplanes/Micro- Ap3, Ap4, Mm1, Mm3, Mm4, Mm5,
phiUW monospora: 51 Aeromonas: 43, 44RR2.8t, 65, Aeh1 Aeromonas
hydrophila: PM1 Agrobacterium: PIIBNV6, PS8, psi, PT11 Alcaligenes:
8764, A5/A6, A6 Alteromonas: PM2 Amycolatopsis: W11, W2, W4, W7
Bacillus: 1A, alpha, AP50, BLE, F, G, GA-1, II, IPy-1, mor1, MP13,
MP15, .o slashed.105, .o slashed.29 (phi 29), .o slashed.NS11,
PBP1, PBS1, SP10, SP15, SP3, SP8, SPP1, SP.beta., SPy-2, SST, type
Bacillus subtilis 168, W23, SP50, W23, SP01 Bdellovibrio: MAC-1,
MAC-2, MAC-4, MAC-5, MAC-7 Brucella: Tb Caulobacter: .o
slashed.Cb12r, .o slashed.Cb2, .o slashed.Cb23r, .o slashed.Cb4, .o
slashed.Cb5, .o slashed.Cb8r, .o slashed.Cb9, .o slashed.CP18, .o
slashed.CP2, .o slashed.Cr14, .o slashed.Cr28 Cellulomonas: O11,
O13, O2, O3, O5, O6, O8 Chlamydia: 1 Chlamydia psittaci: .phi.CPG1
Clostridium: Ce.beta., F1, HM2, HM3, HM7 Coryneforme 7/26, A, A19,
AN25S-1, Arp, AS-1, BL3, CONX, MT, N1, .o slashed.A8010, S-6(L),
.beta., Cyanobacteria: A-4(L), AC-1, LPP-1, S-2L, S-4L, SM-1 E.
coli, (O157): P1, T1, Tula, Tulb, Tull E. coli: 1.o slashed.3, 1.o
slashed.7, 1.o slashed.9, 2D/13, Ae2, alpha10, alpha3, BE/1, BF23,
dA, delta1, delta6, d.o slashed.3, d.o slashed.4, d.o slashed.5,
Ec9, eta8, f1, fd, G13, G14, G4, G6, HK022, HK97, HR, lambda, M13,
M13mp18, M20, MM, MS2, Mu, O1, .o slashed.80, .o slashed.A, .o
slashed.R, .o slashed.X174, PA-2, P1, P1D, P2, P22, Q.beta., R17,
S13, St-1, T1, T2, T3, T4, T5, T6, T7, WA/1, WF/1, WW/1, zeta3,
ZG/2, ZJ/2 E. coli R1: C21 E. coli O8: omega 8 E. coli (K12): U3
Enterobacter: chi, FC3-9, .mu.2, 01, 11F, 121, 1412, 3, 3T+, 50,
5845, 66F, 7480b, 8893, 9, 9266, a1, alpha15, b4, B6, B7, Beccles,
BZ13, C-1, C16, C2, C-2, DdVl, Esc-7-11, f2, fcan, FI, Folac, fr,
GA, H, H-19J, I2-2, Ialpha, ID2, If1, If2, Ike, JP34, JP501, K19,
KU1, M, M11, M12, MS2, NL95, .o slashed.92, .o slashed.l, .O
slashed.ii, Omega8, pilHalpha, PR64FS, PRD1, PST, PTB, R, R17, R23,
R34, sd, SF, SMB, SMP2, SP, .beta., ST, tau, tf-1, TH1, TW18, TW28,
Vill, VK, W31, X, Y, ZG/1, ZIK/1, ZJ/1, ZL/3, ZS/3 Klebsiella
pneumoniae: AP3, C3: Lactobacillus: 1b6, 223, fri, hv, hw222a, .o
slashed.FSW, PL-1, y5 Lactococcus lactis: 1, 643, c2, kh, ml3,
P008, P127, 1358, 1483, 936, 949, BK5-T, c2, KSY1, P001, P008,
P107, P335, PO34, PO87 Leuconostoc: pro2 Listeria: 4211,
Methanothermobacter: psi M2 (.psi.M2) Micrococcus: N1, N5
Mollicutes: Br1, C3, L3 Mycobacterium: I3, lacticola, Leo, .o
slashed.17, R1-Myb Nocardia/Rhodococcus/ N13, N18, N24, N26, N36,
N4, N5 Gordona: Nocardioides: X1, X10, X24, X3, X5, X6, D3, D4,
Pasteurella: 22, 32, AU, C-2 Promicromonospora: P1, P2, P3, P4
Pseudomonas aeruginosa: Phi CT, phi CTX, PB-1 Pseudomonas: 12S, 7s,
D3, F116, gh-1, gh-1, Kf1, M6, .o slashed.1, .o slashed.KZ, .o
slashed.W-14, Pf1, Pseudonocardia: W3 Rhizobium: 2, 16-2-12, 2,
317, 5, 7-7-7, CM1, CT4, m, NM1, NT2, .o slashed.2037/1, .o
slashed.2042, .o slashed.gal-1-R, WT1 Saccharomonospora: Mp1, MP2
Saccharothrix: W1 Salmonella: epsilon15, Felix 01, 16-19, 7-11,
H-19J, Jersey, N4, SasL1, Vil, ZG/3A, San21 Salmonella typhimurium:
A3, A4, P22 Spiroplasma: 4, C1/TS2 Sporichthya: Sp1 Staphylococcus:
107, 187, 2848A, 3A, 44AHJD, 6, 77, B11-M15, Twort Streptococcus:
182, 2BV, A25, A25-24, A25-omega8, A25-PE1, A25-VD13, CP-1, Cvir,
H39 Streptomyces: P23, P26, phi A. streptomycini III, phi8238,
phiC31, S1, S2, S3, S4, S6, S7, SH10 Terrabacter: Tb1, Tb2
Tsukamurella: Ts1 Vibrio: 06N-22P, 06N-58P, 06N-58P, 4996,
alpha3alpha, I, II, III, IV, kappa, nt-1, OXN-100P, OXN-52P, v6,
Vf12, Vf33, VP1, VP11, VP3, VP5, X29 Xanthomonas: Cf, Cf1t, RR66,
Yersinia: 8/C239, phiYeO3-12, YerA41
[0031] If single phage proteins instead of bacteriophages are used,
there is an advantage because in this case the properties of a
single protein instead of a complex of proteins and nucleic acids
may be used. Phage proteins are very stable (Burda et al.,
Biological Chemistry 2000. 381, 255-258); the stability of a single
protein is much easier to control than the stability of a protein
complex. In comparison to complete phages, it is important that
they are easier to modify (genetically, but also chemically), for
example the introduction of tags. Moreover, the use of phage
proteins is an advantage in specific connecting methods, i.e. the
isolation of nucleic acids (plasmid DNA, RNA, genomic DNA), because
compared to the use of complete phages no nucleic acid
contamination is possible.
[0032] Preferred are phage tail proteins from the family of
myoviridae, of podoviridae, and siphoviridae, particularly short
phage tail proteins, particularly the short phage tail proteins of
the even-numbered T-phages, for example T4, T2, or K3, particularly
the bacteriophage tail proteins p12 from T4, p12 from T2 (GenBank
Accession Number X56555), p12 from K3 (cf. Burda et al., 2000,
Biol. Chem., Vol. 381, pp.255-258) or the bacteriophage tail
proteins from the phages Felix 01, P1, or PB1. As an example, the
short bacteriophage tail proteins of the phages T4 (p12) and from
P1 bind to coliformes, the short phage tail protein from Felix 01
binds to salmonellas, and the short phage tail protein from PB1
binds to pseudomonads.
[0033] Phage tail proteins like p12 or P22 tailspike protein
display a high stability over proteases, detergents, chaotropic
agents, for example urea or guanidinium hydrochloride, or higher
temperatures (Goldenberg, D. und King, J.; Temperature-sensitive
mutants blocked in the folding or subunit assembly of the
bacteriophage P22 tail spike protein. II. Active mutant proteins
matured at 30.degree. C., 1981, J. Mol. Biol. 145, 633-651. Miller,
S., Schuler, B. und Seckler, R.; Phage P22 tailspike: Removal of
headbinding domain unmasks effects of folding mutations on
native-state thermal stability, 1998, Prot. Sci. 7, 2223-2232.;
Miller, S., Schuler, B. und Seckler, R.; A reversibly unfolding
fragment of P22 tailspike protein with native structure: The
isolated .beta.-helix domain, 1998, Biochemistry 37, 9160-9168.;
Burda et al., 2000, Biol. Chem., Vol. 381, pp. 255-258). The
removal of the phage head and phage base plate binding region,
respectively, of these proteins may reduce a potentially existing
aggregation sensitivity. Interestingly, the single domains and
subunits, respectively, of these proteins are significantly less
stable than the intact or only marginally reduced trimers (Miller
et al., Prot. Sci. 1998; 7: 2223-2232. Phage P22 tailspike protein:
Removal of head-binding domain unmasks effects of folding mutations
on native-state thermal stability.; Miller-S, et al., Biochemistry
1998; 37: 9160-9168. A reversibly unfolding fragment of P22
tailspike protein with native structure: The isolated .beta.-helix
domain). Furthermore the single domains and subunits, respectively,
are presumably hardly stable and functionally expressable: phage
tail proteins and virus receptor proteins are often available as
intensely drilled trimers, which has been shown in crystallographic
experiments wherein the C-terminus may fold back, which is a
mechanism possibly providing an additional protection against
proteases (Mitraki A, Miller S, van Raaij M J. Review: conformation
and folding of novel Beta-structural elements in viral fibre
proteins: the triple Beta-spiral and triple Beta-helix. J Struct
Biol. 2002 137(1-2):236-247), Moreover, these proteins exist in the
native condition as homotrimers. The trimers contribute with three
binding sites to a stronger binding of bacteria by an increase of
the avidity.
[0034] With the even-numbered T-phages (T4, T2, K3) as an example,
the binding mechanism of the bacteriophage proteins to the single
bacteria should be clarified. In this genus, there are two
components on the host side which are recognised by the phages:
firstly a surface protein specific for individual phages, secondly
the lipopoysaccharide (LPS) which is possessed by all gram-negative
bacteria in a modified form on their outside and is orientated to
the envrionment. The long tail fibres of the even-numbered T-phages
play a role in the specific recognition of the host bacteria,
whereas the LPS serves as a receptor for the short tail fibres. It
is known from the phage T4 from E. coli that the specific
interaction with the host bacterium mediated by the long tail
fibres will become irreversible as soon as the short tail fibres
have been bound to the bacteria surface. The short tail fibre is
not responsible for the correct specificity within the host
bacteria genus and therefore may be replaced between the different
even-numbered T-phages.
[0035] Bacteriophage tail proteins may easily be recombinantly
produced in large numbers and may be purified using appropriate
tags or simple chromatographic standard separation methods. Phages
as well as host strains are largely commercially available via
strain collections or may be isolated by simple means. In the
method of the present invention, however, not only the naturally
occurring bacteriophage tail proteins may be used, but also their
variants. The variants as used in the present invention means that
the bacteriophage tail proteins exhibit an altered amino acid
sequence. Said variants may be obtained by screening of the
naturally occurring variants or by random mutagenesis or targeted
mutagenesis, but also by chemical modification. The bacteriophage
tail proteins used in the method of the present invention may be
adapted by a targeted or random mutagenesis in their host
specificity and their binding behaviours, respectively, to the
support structures. By means of the mutagenesis, mutations are
introduced which may be amino acid additions, deletions,
substitutions, or chemical modifications. These mutations produce
an alteration of the amino acid sequence in the binding region of
the phages or phage proteins, with the intention to adapt the
specificity and binding affinity to the experimental requirements,
for example to enhance the binding of the bacteria to the isolated
phage proteins or to make their binding irreversible, to enhance
the washing options. Moreover, a genetic or biochemical
modification of the phage proteins may be performed with the
intention optionally to switch off the present enzymatic activity
to improve the binding or make the binding irreversible.
[0036] For binding purposes of the bacteria and/or cell components
to be purified to the bacteriophages and/or bacteriophage tail
proteins in the two-step method, the sample, for example an
overnight culture, is contacted with the bacteriophages and/or
bacteriophage tail proteins and is preferably incubated. The
incubation occurs at a temperature in the range of 4.degree. C. to
90.degree. C., preferably at a temperature in the range of
4.degree. C. to 45.degree. C., more preferred at a temperature in
the range of 15.degree. C. to 37.degree. C., furthermore preferred
at a temperature in the range of 20.degree. C. to 37.degree. C., in
particular at RT, for up to 6 hours, preferably up to 4 hours, more
preferred 2 hours, in particular 1 hour, in particular preferred
1-20 minutes, exceptionally preferred 3-5 minutes. For example, the
incubation can occur for 2 to 120 minutes at 4.degree. C. to
37.degree. C., preferably for 20 to 30 minutes at 25.degree. C. to
37.degree. C., preferably more preferred for 3-5 minutes at
37.degree. C.
[0037] The sample is contacted with solid supports subsequently and
incubated. Solid supports may be, for instance, magnetic particles
(paramagnetic or ferromagnetic), glass particles, agarose
particles, luminex particles, reactions tubes, or microtiter
plates.
[0038] In case of using magnetic particles, they were subsequently
added to the sample. The magnetic particles bind the
bacteriophage/bacteriophag- e protein-bacteria/cell component
complex, which is then easily separated from the sample by using
magnetic means, and which may then be purified. The magnetic means
may be positioned at the outside of the container and either may be
switched on for the enrichment so that the magnetic particles are
collected at the container wall, or may slide along the outside
wall of the container so that the magnetic particles are collected
e.g. at the bottom of the container. The enrichment with a
permanent magnet is preferred. The magnetic means may also immerse
into the container and the sample so that the magnetic particles
deposit at the magnetic means (the magnetic means may be covered by
a pipette tip or a comparable disposable). In comparison to
centrifugation or filtration techniques, the bacteria are subject
to only minimal shear rates and therefore may be enriched with high
yield in an active/living manner, if required. The easy handling
facilitates easy and fast buffer/solution changes and may both
easily be performed on a large scale, and well automated.
[0039] The magnetic particles exhibit a diameter allowing the
binding of a sufficient amount of cells or cell components per
particle. Preferably the magnetic particles exhibit a diameter in
the range of about 0.5 to about 4 .mu.m, in particular in the range
of about 0.5 to about 2 .mu.m, more preferred in the range of about
0.8 to about 1.8 .mu.m, most preferred about 1 .mu.m.
[0040] The binding of the bacteriophage/bacteriophage
protein-bacteria/cell component complexes to the solid supports,
for example magnetic particles, preferably occurs via appropriate
coupling groups, in particular polypeptides and/or low molecular
substances. These polypeptides may also be antibodies, lectins,
receptors or anticalins specific for the bacteriophages and/or
bacteriophage proteins. Furthermore, the bacteriophages and/or
bacteriophage proteins may be coupled to low molecular substances,
e.g. biotin, to bind to polypeptides, e.g. streptavidin, via these
low molecular substances wherein the polypeptides may be
immobilised to the support. Instead of biotin, the so-called
strep-tag (Skerra, A. & Schmidt, T. G. M. Biomolecular
Engineering 16 (1999), 79-86) may be used, which is a short amino
acid sequence and binds to Streptavidin. Furthermore, the his-tag
may be used, which may bind to a support material via bivalent ions
(zinc or nickel) or an antibody which is specific for the his-tag
(Qiagen GmbH Hilden, Germany). The strep-tag as well as the his-tag
are preferably bound by means of DNA recombination technology to
the recombinantly produced bacteriophage proteins. This coupling
may occur in a directed manner, e.g. to the N- or C-terminus. Since
particularly in the two-step method a high binding constant is
essential for an effective enrichment, the coupling combination of
biotin/streptavidin with a kD of .about.10.sup.-15M (Gonzales et
al. J. Biol. Chem., 1997, 272 (17), pp. 11288-11294) is preferred
in particular. It was shown that this non-covalent binding
combination works better than the available antibodies, anticalins,
receptors and lectins.
[0041] For a binding of the complex the magnetic particles are
contacted with the bacteriophage/bacteriophage
protein-bacteria/cell component complex and are preferably
incubated. The incubation occurs at a temperature in the range of
4.degree. C. to 90.degree. C., particularly in the range of
4.degree. C. to 45.degree. C., more preferred at a temperature in
the range of 15.degree. C. to 37.degree. C., particularly preferred
at a temperature in the range of 20.degree. C. to 37.degree. C., in
particular at RT, for up to 6 hours, preferably up to 4 hours, more
preferred 2 hours, in particular 1 hour, in particular preferred
1-20 minutes, exceptionally preferred 3-5 minutes. For example, the
incubation can occur for 2 to 120 minutes at 4.degree. C. to
37.degree. C., preferably for 20 to 30 minutes at 25.degree. C. to
37.degree. C., preferably more preferred for 3-5 minutes at
37.degree. C.
[0042] The method of the present invention is performed with other
solid supports which may be added to the sample in the analogous
manner. The single incubation conditions and separation steps have
to be adapted for the different solid supports accordingly. This
may easily be performed in test series and does not require any
further explanation for the skilled artisan.
[0043] Alternatively, the two-step method may be performed in
accordingly coated solid supports which may not be added to the
sample, but wherein the sample is added onto or into the solid
support, e.g. a microtiter plate or a reaction tube. For this
purpose, the sample is added after step a), e.g. into the
respective wells of the microtiter plate, and is incubated there,
particularly for about 20-30 minutes with the other conditions
remaining as described above. The wells of the microtiter plate or
the inner walls of a reaction tube may exhibit the same coatings as
described above for the magnetic particles.
[0044] The enrichment and purification, respectively, of the
bacterial cells and/or cell components may also be performed in a
method comprising the following steps (one-step method):
[0045] a) contacting a sample containing bacterial cells and/or
cell components with a magnetic support on the surface of which
bacteriophages and/or bacteriophage proteins are applied,
preferably with an incubation of about 3-60 minutes,
[0046] b) separating the magnetic support with the bacterial cells
and/or cell components bound to it from the sample.
[0047] The methods according to the present invention (one-step and
two-step method) may be used, for example, as an alternative for
centrifugation and thus for the first time allows the automated
purification of bacterial cells. This for the first time enables
the automation of, e.g., the genome analysis, i.e. from the
inoculation of the bacterial cultures to the determination of the
sequence. Furthermore, the method of the present invention may be
used, for example, to isolate cell components, particularly of
lipopolysaccharides, endotoxines or exopolysaccharides.
[0048] The following embodiments of the coupling or immobilisation
of bacteriophages and/or bacteriophage proteins to the magnetic
particles (one-step method) apply accordingly to the coupling or
immobilisation of bacteriophages and/or bacteriophage proteins and
polypeptides to the solid supports (two-step method). The coating
of the solid supports with the previously described polypeptides or
the bacteriophages and/or bacteriophage proteins may occur in a
different manner.
[0049] The bacteriophages and/or bacteriophage proteins may be
fixed to the solid supports via covalent coupling. This allows a
very tight binding to the support and thus the application of
severe washing conditions for the washing of the cells which is
possibly required for a further processing of the enriched cells.
The coupling of the bacteriophages and/or bacteriophage proteins
via adsorption is a very simple and cost-effective method. One-step
as well as two-step methods are possible by means of coupling the
bacteriophages and/or bacteriophage proteins via
biotin/streptavidin or comparable ligand/receptor systems. The
streptavidin used in this approach may be fixed via adsorption, as
well as via chemical coupling. A functional immobilisation is
important in the coating method, that means that despite their
binding to the solid supports, the bacteriophages and/or
bacteriophage proteins exhibit structures which are accessible to
bacteria.
[0050] The bacteriophages and/or bacteriophage proteins may be
coupled via covalent coupling to support materials which have
already been activated by the manufacturers, for instance to
magnetic particles by Merck, Estapor, etc. via standard conditions,
for example --NH.sub.2 via cyanuryl chloride (Russina Chemical
Rev., 1964, 33: 92-103), or --COO-- via EDC
(1-Ethyl-3'[3'Dimethylaminopropyl]carbodiimid) (Anal. Biochem.
1990, 185: 131-135). Moreover, the solid supports may be activated
directedly using appropriate methods. Furthermore, the coupling may
occur via maleimide or iodoacetyl spacer to, for instance, a
N-terminal introduced cystein.
[0051] The immobilisation of the bacteriophages and/or
bacteriophage proteins to the support material via adsorption may
be performed by incubation of a bacteriophage and/or bacteriophage
protein solution in aqueous buffer, for instance 100 mM Tris pH
7.3, or 100 mM sodium phosphate pH 7.5, PBS (10 mM sodium phosphate
pH 7.4, 150 mM sodium chloride) for several hours or overnight at
4.degree. C. to 45.degree. C., preferably at 15.degree. C. to
37.degree. C., more preferred at 20.degree. C. to 37.degree. C., in
particular preferred at 37.degree. C. or RT, in particular
preferred at 30.degree. C. to 65.degree. C. for 2-4 hours. The
coating solution is discarded after the adsorption and the support
structure is stored in aqueous, optionally in buffered
solution.
[0052] A further aspect of the present invention is a solid
support, in particular a magnetic particle or a microtiter plate,
either coated with bacteriophages and/or bacteriophage proteins, or
coated with polypeptides directed against bacteriophages and/or
bacteriophage proteins. These polypeptides may be antibodies,
lectins, receptors or anticalins specific for the bacteriophages
and/or bacteriophage proteins. The solid supports may be coated
furthermore with streptavidin.
[0053] A further aspect of the present invention are bacteriophage
proteins coupled with so-called tags, for example the strep- or the
his-tag, particularly to the 3'- or 5' terminus, more preferred to
the 5' terminus. The coupling or linking of the tags with the
bacteriophage proteins via DNA recombination technology is
preferred. The production of the nucleic acid, comprising the
sequence of the bacteriophage protein and the tag, and the
production of the expression product are state of the art and there
is no need to explain the production in detail at this point. A
further aspect of the present invention is the nucleic acid
sequence coding the bacteriophage protein together with the strep-
or the his-tag. A p12 protein from the bacteriophage T4 is a
particularly preferred bacteriophage protein modified with the
strep- or the his-tag, however, all other bacteriophage proteins of
the listed bacteriophages from the above table are also
preferred.
[0054] A further aspect of the present invention are bacteriophage
proteins with a tag exhibiting a surface-exposed cysteine for the
specific, directed biotinylation, e.g. the tags according to SEQ ID
NOs: 5, 6 or 7. One example for a p12 with a tag is the amino acid
sequence depicted in SEQ ID NO: 8. Preferred is a p12 with a tag,
in particular with a tag with a surface-exposed cysteine, in
particular a p12 with a tag according to SEQ ID NOs: 6 and 7. In
addition, said directed biotinylation may be mediated by an
appropriate spacer or linker. Furthermore, the present invention
relates to the amino acid sequences according to SEQ ID NOs: 5, 6
and 7. Furthermore, the present invention relates to nucleic acids
coding for the amino acid sequences according to SEQ ID NOs: 5, 6
and 7.
[0055] A further aspect of the present invention relates to a kit
for the enrichment of bacterial cells and/or cell components,
comprising the solid supports according to the present invention,
for example the magnetic particles, glass particles, agarose
particles, reaction tubes or microtiter plates as well as the
solutions including the test reagents necessary for the enrichment
of the bacteria and/or cell components.
[0056] The kit for the enrichment with magnetic particles includes
in particular a stabilised solution of a p12-variant with a
cysteine residue for the directed biotinylation introduced at the
N-terminus, for example NS-T4p12 (or T4p12bio) 1 mg/ml in 100 mM
Tris HCl pH8, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20, supplement
with a protease inhibitor mixture (Sigma) as a solution (preferred
storage at 4.degree. C.) or as a lyophilisate. Furthermore, the kit
includes a particle solution consisting of streptavidin- or
streptactin-coated magnetic particles in a stabilising solution
(PBST with sodium azide 0.005%).
[0057] The kit for an enrichment with microtiter plates includes in
particular a stabilised solution of a p12-variant with a cysteine
residue for the directed biotinylation at the N-terminus, for
example NS-T4p12 (or T4p12bio) 1 mg/ml in 100 mM Tris HCl pH8, 150
mM NaCl, 1 mM EDTA, 0.05% Tween 20, supplement with a protease
inhibitor mixture (Sigma) as a solution (preferred storage at
4.degree. C.) or as a lyophilisate. The kit furthermore includes a
streptavidin- or streptactin-coated microtiter plate.
[0058] The following examples illustrate the invention and are not
to be understood as limiting. If not indicated otherwise, molecular
biological standard methods have been used, as for example
described by Sambrook et al., 1989, Molecular cloning: A Laboratory
Manual 2. Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0059] 1. The Purification of Wild Type T4p12 was Carried Out
According to the Method Described in Burda, M. R. & Miller, S.
Eur. J. Biochem. 1999, 265 (2), 771-778.
[0060] 2. Construction of p12 with a N-Terminal Strep-Tag
(N-Strep-p12):
[0061] The nucleotide sequence of the strep-tag (U.S. Pat. No.
5,506,121) was introduced to the 5' terminus of the T4p12 gene via
PCR. For the 5' terminus of the p12 gene a primer was constructed
(5'-GAA GGA ACT AGT CAT ATG GCTAGC TGG AGC CAC CCG CAG TTC GAA AAA
GGC GCC AGT MT MT ACA TAT CM CAC GTT-3'), (SEQ ID NO:1) including
the nucleotide sequence of the strep-tag at its 5' terminus
(printed in italics in the sequence) and having a restriction
recognition sequence (NdeI, underlined in the sequence) in such a
manner that the gene may be inserted in the correct reading frame
into the expression plasmid. A primer was constructed was
constructed for the 3' terminus of the p12 gene introducing a BamH1
restriction recognition sequence (printed in italics in the
sequence) behind the p12 gene (5'-ACG CGC AAA GCT TGT CGA CGG ATC
CTA TCA TTC TTT TAC CTT MT TAT GTA GTT-3'), (SEQ ID NO:2). The PCR
was performed with 40 cycles (1 min 95.degree. C., 1 min 45.degree.
C., and 1 min 72.degree. C.). The PCR preparation was cleaved with
the restriction endonucleases NdeI and BamH1 and the desired
fragment was inserted in the NdeI and BamH1 site of the expression
plasmid pET21a (Novagen, Merck Eurolab, Darmstadt, DE) after size
directed separation via an agarose gel and elution from the gel.
The sequence of the N-strep-p12 gene was verified by DNA
sequencing. The further steps to the plasmid pNS-T4p12p57 were
performed as described by Burda, M. R. & Miller, S. (Eur. J.
Biochem. 1999, 265 (2), 771-778). The plasmid pNS-T4p12p57 was then
transformed into the expression strain BL21 (DE3) (Novagen, Merck
Eurolab, Darmstadt, DE).
[0062] 3. Introduction of a N-Terminal Cysteine Residue in
N-Strep-p12 (N-Strep-S3C-p12 and N-Strep-S14C-p12):
[0063] The introduction of a N-terminal cysteine residue (bold
type) was performed as described under 2. above with two new
primers for the 5' terminus being constructed. The primer 5'-GM GGA
ACT AGT CAT ATG GCT TGT TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT
MT MT ACA TAT CM CAC GTT-3' (SEQ ID NO:3) was used for the
N-strep-S3C-p12, and the primer 5'-GM GGA ACT AGT CAT ATG GCTAGC
TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC TGT MT AAT ACA TAT CM CAC
GTT-3' (SEQ ID NO:4) was used for the N-strep S14C-p12.
[0064] 4. Purification of N-Strep-p12 Protein:
[0065] The E. coli strain BL21 (DE3) with the plasmid pNS-T4p12p57
was cultured in 21 shaking cultures (LB medium with ampicillin 100
.mu.g/ml) up to an OD600 of 0.5-0.7 at 37.degree. C. and the
expression of the N-strep-p12 protein was induced by adding 1 mM
IPTG (Isopropyl-.beta.-thiogalactopyranoside). The cells were
harvested after incubation at 37.degree. C. for 4 hours. Harvested
cells from a 10 l culture were resuspended in 50 ml sodium
phosphate, 20 mM pH 7.2, 2 mM MgSO.sub.4, 0.1 M NaCl, broken up by
a triple French press treatment (20,000 psi), and were subsequently
centrifuged for 30 minutes at 15,000 rpm (SS34). After washing it
twice in the same buffer, the N-strep-p12 protein was extracted
from the pellet by stirring for 30 minutes in 40 mM Tris HCl pH
8.0, 10 mM EDTA. The preparation was centrifuged for 30 minutes at
15,000 rpm (SS34) and the supernatant with the separated NS-p12 was
stored at 4.degree. C. The extraction was repeated twice and the
combined supernatants were applied to a streptactin-affinity column
(15 ml) equilibrated with buffer "W" (100 mM Tris HCl pH 8, 1 mM
EDTA, 150 mM NaCl) (IBA GmbH, Gottingen, D E). After washing with 5
volumes of the column with buffer "W", the column was eluted with 3
volumes of buffer "W" with 2.5 mM desthiobiotin in buffer "W".
After repeated dialysis with "W" and concentration, the
concentration and purity of N-strep-T4p12 was determined via
SDS-PAGE and UV Spectroscopy (Burda et al. 1999). In this way,
about 100 mg N-strep-T4p12 were purified from a 10 l culture.
2 Name Sequence of the tag Nstrep-p12 MASWSHPQFEKGAS SEQ ID NO: 5
Nstrep-p12-S3C MACWSHPQFEKGAS SEQ ID NO: 6 Nstrep-p12-S14C
MASWSHPQFEKGAC SEQ ID NO: 7
[0066] 5. Alternative Purification of p12:
[0067] The cell pellet (from a 10 l culture, BL21 (DE3),
transformed with the plasmid pNS-T4p12p57 or pT4p12p57) was
resuspended in buffer 1 (10 mM sodium phosphate pH 9, 500 mM NaCl,
4 mM MgCl.sub.2) and broken up via French press (as described under
3.). Subsequently the material was centrifuged at 20,000 rpm (SS34)
for 45 minutes and the pellet was resuspended (i.e. washed) in
about 25 ml of buffer 1. This washing step was repeated twice and
the pellet was extracted with 25 ml of buffer 2: (50 mM NaPi pH
5,100 mM NaCl, 25 mM EDTA). The resuspended pellet was stirred for
the extraction for 60 minutes at RT and afterwards centrifuged
(20,000 rpm (SS34) for 45 minutes). Said extraction was repeated
twice if required. Afterwards, the supernatants of the extraction
were combined and applied directly to an anion exchange column
(ResoureceS, Pharmacia). 15 mM sodium hydrogen phosphate, 15 mM
sodium formiate, 30 mM sodium acetate, pH 5.0, 50 mM NaCl were used
as a running buffer. The elution occurred via a linear salt
gradient of 50 mM NaCl to 60 mM NaCl in the running buffer. The
purified p12 was subsequently dialysed for storage against 50 mM
Tris pH 8.5, 150 mM NaCl, 5 mM EDTA or PBS, frozen in aliquots in
N.sub.2, and stored at -20.degree. C.
[0068] 6. Biotinylation of p12:
[0069] For the biotinylation of the p12 protein, either
EZ-Link.TM.Sulfo-NHS-LC-LC-Biotin or EZ-Link.TM.TFP-PEO-Biotin by
Pierce, USA, was used or the biotinylation was performed according
to the methods of the manufacturer. About 30 molecules of biotin
per p12-trimer were used for the biotinylation. The coupling of
biotin was subsequently verified with a HABA-assay (Savage M D,
Mattson G, Desai S, Nielander G W, Morgensen S, and Conklin E J,
1992, Avidin-Biotin Chemistry: A Handbook, Pierce, Ill.) and
quantified. Finally, an average of 5-10 molecules of biotin per
p12-trimer were bound.
[0070] 7. For the Biotinylation of the N-Terminal Introduced
Cysteine, EZ-Lin.TM.PEO-Maleiimid-Biotin and
EZ-Link.TM.PEO-Liodoacetyl-Biotin by Pierce, USA, were Used
According to the Instructions of the manufacturer. The Reaction was
Verified as Described under 5. Above.
[0071] 8. Biotinylation of the Bacteriophage T4:
[0072] The bacteriophage was purified according Bachrach U and
Friedmann A (1971) Practical procedures for the purification of
bacterial viruses, Appl. Microbiol 22: 706-715. The purified phage
was dialysed against PBS and labelled with EZ-Link.TM.Sulfo
-NHS-LC-LC-Biotin or EZ-Link.TM.TFP-PEO-Biotin by Pierce, USA,
according to the instructions of the manufacturer in a ratio of
100-100,000 biotin molecules per phage.
[0073] 9. p12-Dependent Harvest of E. Coli According to the
One-Step Method and the Two-Step Method (FIG. 1):
[0074] In the binding step according to the one-step method (VC)
the N-strep-p12 protein was incubated for 1 hour with the magnetic
streptavidin beads (10 .mu.g protein/50 .mu.l 1% beads) and the
beads were subsequently washed three times with PBST. For the cell
binding step, 200 .mu.l of an E. coli overnight culture diluted 1
to 10 in LB (about 1.times.10.sup.8 cells/ml) per well in a
microtiter plate were mixed with 10 .mu.l of the N-strep-p12 coated
beads and incubated for different periods of time at RT (FIG. 1).
The beads were concentrated subsequently with the bound cells via a
magnetic separator (Bilatec A G, Mannheim, D E) for 3-5 minutes at
the walls of the wells. The beads were washed three times with PBST
and the .beta.-galactosidase activity (Apte S C et al., 1995, Water
res. 29, 1803-06) of the cells attached to the beads were
determined subsequently. For the binding step according to the
two-step method, a 1 to 10 dilution of an E. coli overnight culture
(about 1.times.10.sup.8 cells/ml) were incubated for 1 hour (in
further approaches for 1 minute, 3 minutes, 10 minutes, 30 minutes)
with the N-strep-p12 protein (10 .mu.g protein/ml cell suspension)
at RT. Subsequently, 200 .mu.l of the protein-cell-mixture were
added to 10 .mu.l of 1% magnetic streptavidin beads and incubated
at RT for the times given in FIG. 1. The determination of the bound
cells was performed according to the one-step method.
[0075] 10. T4-Dependent Binding of E. Coli According to the
One-Step Method (VC) and the Two-Step Method (VIK) (FIG. 2)
[0076] In the binding step according to the one-step method (VC),
the biotinylated phage T4 (100 biotin molecules/phage) were bound
for 1 hour to 1% streptavidin beads (10.sup.10 PFU/ml 1% beads) and
the beads were washed three times with PBST subsequently. After 2
hours of the cell binding step (25 .mu.l phage beads/ml cell
suspension), the beads were washed and the bound E. coli were
determined via the .beta.-galactosidase activity (the data are
given in relative units). In the binding step according to the
two-step method (VIK), the cells were incubated with the
biotinylated phages for 1 hour (2.5.times.10.sup.8 PFU/ml cell
suspension) and the mixture was incubated subsequently for 2 hours
with the streptavidin beads (25 .mu.l 1% beads/ml cell suspension).
The further steps were performed according to the one-step method.
During the continuous incubation of phage and bacteria, the
antibiotics Rifampicin (25 .mu.g/ml), Chloramphenicol (25 .mu.g/ml)
and Tetracycline (2 .mu.g/ml) were added to the medium.
[0077] 11. Harvest of E. Coli Cells According to the Two-Step
Method From Different Growth Media (FIG. 3):
[0078] The E. coli strains LE392 and JM83 were grown overnight in
the respective media. N-strep-p12 (10 .mu.g/ml cell suspension)
were added to 200 .mu.l of cell suspension. 10 .mu.l 1%
streptavidin beads were added after 5 minutes of incubation at RT,
mixed by pulling it three times with a pipette, and were incubated
a further 5 minutes at RT. Subsequently, the bacteria beads
complexes were removed by means of the above described magnetic
separator for 3-5 minutes and the cells remaining in the
supernatant were determined via the scattering of the supernatant
at 600 nm.
[0079] 12. Plasmid Isolation of E. coli After Cell Harvest via the
Two-Step Method (FIG. 4).
[0080] 300 .mu.l each of a E. coli overnight culture containing the
plasmid pUC19 were harvested according to the two-step method as
described under example 9. After removal of the cells by means of
the above described magnetic separator, the plasmid was isolated
from the cells in a first method via the solid phase extraction
methods (QIAprep, Qiagen, Hilden, D E) and also via a method using
magnetic beads (Bilatec, Mannheim, D E) according to the
instructions of the manufacturers.
[0081] 13. Enrichment of E. Coli Cells from 10 ml Culture Volume
VIA N-Strep-p12 and via the Biotinylated Bacteriophage T4-Bio
According to the Two-Step Method (FIG. 5):
[0082] For the enrichment with N-strep-p12, 10 ml of E. coli
cultures with 10.sup.9, 10.sup.8 and 10.sup.7 cells per ml were
mixed with 30 .mu.g and 6 .mu.g protein each, respectively, and
scrolled for 1 hour at 37.degree. C. For the enrichment with
T4-bio, 10 ml cultures with 10.sup.9, 10.sup.8 and 10.sup.7 cells
per ml were each added to 10.sup.10 T4-bio phages and 10 .mu.g
Rifampicin/ml and 2 .mu.g Tetracycline/ml and scrolled for 1 hour
at 37.degree. C. Subsequently, 100 .mu.l 1% magnetic streptavidin
beads were added to the preparations and scrolled for 1 more hour.
By means of a magnet (ABgene, Hamburg, DE), the cells bound to the
magnetic beads were separated, washed three times with PBST, and
determined via their .beta.-galactosidase activity.
[0083] 14. Living Harvest of E. Coli via the Two-Step Method with
Biotinylated p12 (FIG. 6):
[0084] E. coli cells (200 .mu.l of a culture) were harvested
according to the two-step method as described in example 9 (10
.mu.g biotinylated p12/ml cell culture and 10 .mu.l 1% streptavidin
beads/ml cell culture) and the beads washed twice with PBST.
Subsequently, the .beta.-galactosidase activity and the growth of
the cells after 2 hours was determined via the scattering at 600 nm
in a photometer.
[0085] 15. Selectivity of the N-Strep-p12-Dependent Harvest (FIGS.
7 and 8):
[0086] 200 .mu.l each of an overnight culture of different E. coli
strains (FIG. 7) as well as of different bacteria strains (FIG. 8)
were harvested according to the two-step method (as described in
example 9). After concentration of the beads in a magnetic
separator, the separated cell amount was determined via the
scattering of the supernatant at 600 nm.
[0087] 16. Selective Binding of E. coli to Magnetic Streptavidin
Beads via T4-Bio and Assay of E. Coli via FITC-Labelled p12 (FIG.
9):
[0088] The p12 protein was labelled with FITC (Molecular Probes,
Leiden, N L) according to the instructions of the manufacturer and
dialysed against PBS. FITC-labelled p12 (5 .mu.g/ml) was added to a
mixed culture (about 10.sup.8-9 cells/ml) from E. coli and Serratia
marcescens. After 5 minutes of dark incubation at RT, 10.sup.9
T4-bio phages and Rifampicin (10 .mu.g/ml), Chloramphenicol (25
.mu.g/ml) and Tetracycline (2 .mu.g/ml) were added. As a control,
non-biotinylated T4 phage was added. After an incubation of 10
minutes, 1% streptavidin beads (10 .mu.l/ml) were added and the
preparations were studied under a microscope (FIG. 9).
[0089] 17. Determination of the Detection Limit for the
N-Strep-p12-Dependent Isolation of E. Coli Cells According to the
Two-Step Method (FIG. 10):
[0090] 300 .mu.l each of dilutions of an E. coli overnight culture
(10.sup.2-10.sup.15 cells/ml) were incubated in microtiter plates
with N-strep-p12 (10 .mu.g protein/ml) for 1 hour.
[0091] Subsequently magnetic streptavidin beads (100 .mu.l 1%
beads/ml) were added, mixed, and the bound cells were pelleted by
means of a magnet (Bilatek, Mannheim, D E). The beads were washed
three times with PBST. The detection of the E. coli cells occurred
via fluorescence and luminescence substrates for the
.beta.-galactosidase.
[0092] 18. Chemical Coupling of T4p12 to Magnetic Beads (FIG.
11):
[0093] 150 .mu.l 1% magnetic beads (EM2-100/40, Merck Eurolab,
France) were washed three times with 10 mM sodium phosphate buffer,
pH 6, resuspended in 40 .mu.l of the buffer. After adding 120 .mu.l
EDC-solution (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30
mg/ml) and incubation for 5 minutes at RT, 160 .mu.l of T4p12
solution (0.7 mg protein/ml 10 mM sodium phosphate buffer, pH 6)
was pipetted to the solution, mixed, and the preparation was
incubated for 2 hours at RT. By adding 1 volume of 0.2 M Tris-HCl,
pH 7, 0.05% Tween 20, the reaction was stopped at 4.degree. C.
overnight. The beads were washed subsequently 4 times with PBST and
adjusted to 1% with PBST. The coupling of p12 to the beads was
determined via a p12-specific antiserum. The binding activity of
the p12 beads was determined via the binding of E. coli cells
according to the one-step method. 3 .mu.l 1% p12 beads were
incubated with 200 .mu.l of a hundredfold diluted E. coli overnight
culture (about 1.times.10.sup.7 cells/ml) for 5 minutes, washed
three times with PBST, and the bound cells were detected via their
.beta.-galactosidase activity (FIG. 11).
[0094] 19. Adsorption of p12 to Different Magnetic Polyvinylalcohol
(PVA) Beads (FIG. 12):
[0095] 200 .mu.l 2% PVA beads (Chemagen, A G, Baesweiler, D E) were
incubated with different amounts of T4p12 (0-5 .mu.g protein/mg
beads) in 100 mM Tris HCl, pH 8, 1 mM EDTA, 200 mM NaCl overnight
at 37.degree. C. The beads were subsequently washed two times with
PBST and resuspended in PBS to give 2%. The functional binding of
T4p12 to the beads was determined via the binding of E. coli cells.
200 .mu.l of an E. coli overnight culture were mixed with 10 .mu.l
of p12 beads and incubated for 5 minutes at RT. After removal of
the bound cells, the scattering of the supernatant was measured at
600 nm and set in relation to the scattering of the E. coli culture
before adding the beads.
Sequence CWU 1
1
8 1 78 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 1 gaaggaacta gtcatatggc tagctggagc cacccgcagt
tcgaaaaagg cgccagtaat 60 aatacatatc aacacgtt 78 2 54 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 2
acgcgcaaag cttgtcgacg gatcctatca ttcttttacc ttaattatgt agtt 54 3 78
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 3 gaaggaacta gtcatatggc ttgttggagc cacccgcagt
tcgaaaaagg cgccagtaat 60 aatacatatc aacacgtt 78 4 78 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 4
gaaggaacta gtcatatggc tagctggagc cacccgcagt tcgaaaaagg cgcctgtaat
60 aatacatatc aacacgtt 78 5 19 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Peptide 5 Met Ala Ser Trp Ser His
Pro Gln Phe Glu Lys Gly Ala Ser Asn Asn 1 5 10 15 Thr Tyr Gln 6 19
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Peptide 6 Met Ala Cys Trp Ser His Pro Gln Phe Glu Lys Gly
Ala Ser Asn Asn 1 5 10 15 Thr Tyr Gln 7 19 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 7 Met Ala Ser
Trp Ser His Pro Gln Phe Glu Lys Gly Ala Cys Asn Asn 1 5 10 15 Thr
Tyr Gln 8 539 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 8 Met Ala Ser Trp Ser His Pro Gln Phe
Glu Lys Gly Ala Ser Asn Asn 1 5 10 15 Thr Tyr Gln His Val Ser Asn
Glu Ser Arg Tyr Val Lys Phe Asp Pro 20 25 30 Thr Asp Thr Asn Phe
Pro Pro Glu Ile Thr Asp Val Gln Ala Ala Ile 35 40 45 Ala Ala Ile
Ser Pro Ala Gly Val Asn Gly Val Pro Asp Ala Ser Ser 50 55 60 Thr
Thr Lys Gly Ile Leu Phe Leu Ala Thr Glu Gln Glu Val Ile Asp 65 70
75 80 Gly Thr Asn Asn Thr Lys Ala Val Thr Pro Ala Thr Leu Ala Thr
Arg 85 90 95 Leu Ser Tyr Pro Asn Ala Thr Glu Ala Val Tyr Gly Leu
Thr Arg Tyr 100 105 110 Ser Thr Asp Asp Glu Ala Ile Ala Gly Val Asn
Asn Glu Ser Ser Ile 115 120 125 Thr Pro Ala Lys Phe Thr Val Ala Leu
Asn Asn Val Phe Glu Thr Arg 130 135 140 Val Ser Thr Glu Ser Ser Asn
Gly Val Ile Lys Ile Ser Ser Leu Pro 145 150 155 160 Gln Ala Leu Ala
Gly Ala Asp Asp Thr Thr Ala Met Thr Pro Leu Lys 165 170 175 Thr Gln
Gln Leu Ala Val Lys Leu Ile Ala Gln Ile Ala Pro Ser Lys 180 185 190
Asn Ala Ala Thr Glu Ser Glu Gln Gly Val Ile Gln Leu Ala Thr Val 195
200 205 Ala Gln Ala Arg Gln Gly Thr Leu Arg Glu Gly Tyr Ala Ile Ser
Pro 210 215 220 Tyr Thr Phe Met Asn Ser Thr Ala Thr Glu Glu Tyr Lys
Gly Val Ile 225 230 235 240 Lys Leu Gly Thr Gln Ser Glu Val Asn Ser
Asn Asn Ala Ser Val Ala 245 250 255 Val Thr Gly Ala Thr Leu Asn Gly
Arg Gly Ser Thr Thr Ser Met Arg 260 265 270 Gly Val Val Lys Leu Thr
Thr Thr Ala Gly Ser Gln Ser Gly Gly Asp 275 280 285 Ala Ser Ser Ala
Leu Ala Trp Asn Ala Asp Val Ile His Gln Arg Gly 290 295 300 Gly Gln
Thr Ile Asn Gly Thr Leu Arg Ile Asn Asn Thr Leu Thr Ile 305 310 315
320 Ala Ser Gly Gly Ala Asn Ile Thr Gly Thr Val Asn Met Thr Gly Gly
325 330 335 Tyr Ile Gln Gly Lys Arg Val Val Thr Gln Asn Glu Ile Asp
Arg Thr 340 345 350 Ile Pro Val Gly Ala Ile Met Met Trp Ala Ala Asp
Ser Leu Pro Ser 355 360 365 Asp Ala Trp Arg Phe Cys His Gly Gly Thr
Val Ser Ala Ser Asp Cys 370 375 380 Pro Leu Tyr Ala Ser Arg Ile Gly
Thr Arg Tyr Gly Gly Ser Ser Ser 385 390 395 400 Asn Pro Gly Leu Pro
Asp Met Arg Gly Leu Phe Val Arg Gly Ser Gly 405 410 415 Arg Gly Ser
His Leu Thr Asn Pro Asn Val Asn Gly Asn Asp Gln Phe 420 425 430 Gly
Lys Pro Arg Leu Gly Val Gly Cys Thr Gly Gly Tyr Val Gly Glu 435 440
445 Val Gln Lys Gln Gln Met Ser Tyr His Lys His Ala Gly Gly Phe Gly
450 455 460 Glu Tyr Asp Asp Ser Gly Ala Phe Gly Asn Thr Arg Arg Ser
Asn Phe 465 470 475 480 Val Gly Thr Arg Lys Gly Leu Asp Trp Asp Asn
Arg Ser Tyr Phe Thr 485 490 495 Asn Asp Gly Tyr Glu Ile Asp Pro Ala
Ser Gln Arg Asn Ser Arg Tyr 500 505 510 Thr Leu Asn Arg Pro Glu Leu
Ile Gly Asn Glu Thr Arg Pro Trp Asn 515 520 525 Ile Ser Leu Asn Tyr
Ile Ile Lys Val Lys Glu 530 535
* * * * *